Measurement of Nano particles and Proteins

Ulf Nobbmann, Biophysical Characterization, Malvern Instruments Ltd., UK
Renate Hessemann, Marketing Manager Europe, Malvern Instruments, Germany

Colloid emulsions in their native environment require particle size determination in high concentration since there are concerns about changes in sample morphology upon dilution eg break up of aggregates. For proteins and other samples with very low concentration and for weakly scattering samples high sensitivity is required.
New technologies offer both.

Dynamic Light Scattering (DLS) is a powerful technique for determining the size of sub-micron particles. Conventional instrumentation is limited in terms of the maximum concentration of samples that can be analysed because of multiple scattering effects. Non-invasive back scatter (NIBS) technology not only increases the concentration limits at which DLS can be successfully applied, but also increases the sensitivity of the technique.

Dynamic light scattering
Dynamic light scattering (DLS) is a non-invasive technique for measuring particle size, typically in the sub-micron size range. Particles in suspension undergo random Brownian motion. If these particles are illuminated with a laser beam, the laser light is scattered. The intensity of the scattered light detected at a particular angle fluctuates at a rate that is dependent upon the particle diffusion speed, which in turn is governed by particle size. Particle size data can therefore be generated from an analysis of the fluctuations in scattered light intensity

Concentration limits of DLS
In the past 20 years, DLS has become a routine tool for the measurement of particles less than a micron. However, conventional DLS has its limitations. The sample concentration must be high enough to ensure an adequate signal, but the risk of erroneous results due to multiple scattering (light scattered by one particle undergoing scattering by another) means there are restrictions on the concentration range for which measurements are valid.

NIBs extends the concentration limits
The use of patented noninvasive backscatter technology has overcome these limitations. NIBS is a dynamic light scattering technique incorporating an optical configuration that maximizes the detection of scattered light while maintaining signal quality. This provides the high sensitivity needed for measurement of the size and molecular weight of molecules smaller than 1000 D. It also enables measurement at extremely high concentrations. The use of backscattering, rather than more typically detecting scattered light at a 90o angle, improves the sensitivity and at the same time ensures the smallest possible interference from multiple scattering. Previous backscattering techniques have suffered from drawbacks that include the need for close contact between sample and detector optics, necessitating frequent cleaning of both the measurement cell and the detector. Because NIBS is a non contact technique, cleaning is not necessary.

The range of sample concentrations that can be analyzed successfully is extended by changing the measurement position within the cuvette. This is achieved by moving the focussing lens. For small particles, or samples at low concentrations, it is beneficial to maximize the amount of scattering from the sample and hence a measurement position towards the centre of the cuvette is most effective. Large particles, or samples at high concentrations, scatter much more light and therefore measuring closer to the cuvette wall is preferable as this reduces the chance of multiple scattering.

DLS for protein characterization
One of the most time consuming steps is still the actual crystallisation, the search for the conditions under which the protein under investigation will crystallize. As the screening involves a plethora of various buffer conditions and protein amounts are often limited it has become wide-spread practice to check the suitability of the starting material. A simple light scattering experiment will tell the size and the polydispersity, the non-homogeneity of the starting sample. The measurement is quick, and requires only small volumes, with the intact sample being available for further analysis.

What information does one get out of the technique?
The size itself can be linked to the molecular weight of the protein. While there may be unusual shape effects, many proteins tend to behave like relatively globular molecules. And these may then be expected to behave according to the Mark-Houwink relation: the measured particle size is related to the molecular weight through a power law. When encountering an ‘unknown’ protein, it is a simple matter of comparing its measured size to the expected estimated molecular weight. Thus, the size can predict the oligomeric state of the protein in solution. As the measured size gives an impression of the molecules as it is present in the sample under the current conditions, it provides insight into the actual oligomeric configuration. This, however, requires a reasonable data quality. Very polydisperse samples are not suitable for such advanced data interpretation.
The polydispersity is the width of the size distribution. When many different particle species are present in a measuring volume, the width of the size distribution will turn out to be larger than when all particles are of the same size.

In real life, there seems to be a “natural polydispersity” due to constant interchange with solvent layer molecules and some molecular flexibility. However, when different particle species, such as dimers, trimers, oligomers are present then the polydispersity is markedly higher than for monodisperse single-meric solutions.

The relative polydispersity expressed in percent of the half width of the peak divided by the peak mean in the particle size distribution can vary from a few to a hundred percent. Many proteins show polydispersities below 20% for single species, 20-30% for oligo-species (monomer-dimer, or monomer-tetramer), and above 30% when forming a wide range of different oligomeric states in the buffer under question.

If the question is solubility at different buffer compositions then light scattering is the fastest answer. It provides the particle size (which gives an estimate of the oligomeric state of the protein) and the polydispersity (which shows the homogeneity of the distribution in solution). The ease and speed of the technique has shown advantages in protein crystallization, stability analysis, thermal properties, degradation, self-assembly, in short characterization in solution conditions.

Zetasizer Nano - optimized for Protein characterization
Malvern Instruments’ Zetasizer Nano S combined static and dynamic light scattering instrument is optimized for the characterization of proteins in solution prior to crystallization.
This compact, easy to use system is designed for the rapid delivery of accurate and extensive information that can assist both in the screening of appropriate conditions for protein crystallization and in determining the likelihood of crystals being suitable for structure determination. Not only does the Zetasizer Nano automatically optimize all instrument settings for each sample but custom data reports and graphical data presentations make interpretation easier than ever before.

The Zetasizer Nano enables researchers to detect and quantify aggregation, determine the second virial coefficient to find the “crystallisation sweet spot”, and quantify sample polydispersity to increase the likelihood of successful crystallization.

In addition it enables users to study the effect of temperature on monodispersity, and offers the ability to automate temperature studies, including melting point and thermal denaturation determinations.

It also allows estimation of prolate and oblate axial ratios and Perrin factor, as well as measurement of hydrodynamic diameter and absolute molecular weight.

Size measurements of proteins as small as 0.6 nm and 400 Da can be made in their native environments. As little as 12 microliters of sample is required and the sample is recoverable.